Clostridium Perfringens Extracellular Toxins and Enzymes: 20 and Counting

Under the Microscope

Clostridium perfringens extracellular toxins and enzymes: 20 and counting

Sarah A Revitt-Mills A, Julian I Rood A and Vicki Adams A,B AMonash University, 19 Innovation Drive, Clayton, Vic. 3800, Australia, Tel: +61 3 9902 9139, Fax: +61 3 9902 2222 BEmail: [email protected]

Clostridium perfringens is a Gram-positive, anaerobic bac- toxins (alpha, beta, epsilon and iota)4,9. This typing scheme is now terium that is widely distributed in the environment; it is very much outdated, but it has been useful for classification as the found in soil and commonly inhabits the gastrointestinal different toxinotypes are often associated with specific diseases4,10 tract of humans and animals1,2. The ubiquitous nature of (Table 1). For example, clostridial myonecrosis and human food this bacterium has resulted in it becoming a major cause of poisoning are associated with type A strains, whereas type B, C histotoxic and enteric diseases3. The success of C. perfrin- and D strains are most strongly associated with enteric diseases of gens as both a pathogen and a commensal bacterium lies in livestock4. its ability to produce a large number of potent toxins and extracellular enzymes4. This diverse toxin repertoire results Toxins and toxin gene location in a broad range of diseases including gas gangrene, various The number of characterised C. perfringens toxins is ever enterotoxaemias, food poisoning and necrotic enteritis4–6. increasing; with more than 20 different toxins and enzymes classi- Since 2007, six new toxins have been identified, adding to the fied to date, see Table 13,5,9,11. With a few important exceptions, – ever-increasing range of potential C. perfringens virulence these toxins are encoded on large conjugative plasmids4,10,12 18, determinants. This paper briefly reviews the plethora of which allows for potential toxin gene transfer between different toxins and extracellular enzymes produced by C. perfrin- C. perfringens strains in the gastrointestinal tract and may prolong gens, highlighting their importance in disease and strain disease10. C. perfringens utilises chromosomally encoded toxins, classification as well as introducing the latest additions to such as alpha-toxin and perfringolysin O, during human histotoxic the ever increasing C. perfringens toxin family. infections or human food poisoning (C. perfringens enterotoxin, CPE)3. However, for reasons that are probably related to disease Toxinotype strain classification epidemiology, plasmid-encoded toxins are critical for non-food- Like many clostridial species, the virulence of C. perfringens is borne human gastrointestinal diseases, human enteritis necroticans dependent on the production of toxins7. Not all toxins are produced and gastrointestinal diseases of animals3,10. by any one strain, instead individual isolates vary in toxin carriage and production8. This toxin expression profile forms the basis of a The toxin categories of C. perfringens toxinotype classification scheme; in which strains are classified into The toxins of C. perfringens can be functionally classified into four five toxinotypes (A–E) based on the production of four different broad categories: membrane damaging enzymes, pore-forming

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Table 1. Properties of Clostridium perfringens toxins. Toxin/Enzyme Gene Activity Associated disease Gene location

Alpha-toxin plc or cpa Phospholipase C and CM: humans and animals Chm Sphingomyelinase

Beta-toxin cpb Pore-forming toxin NE in humans and animals Plasmid

Epsilon-toxin etx Pore-forming toxin ET in sheep and goats Plasmid

Iota-toxin iap/ibp Actin-speciﬁc ADP E in sheep and cattle, ET in Plasmid ribosyltransferase rabbits

Enterotoxin cpe Pore-forming toxin C and E in domestic ungulates, Chm or Plasmid FP and GI in humans

Theta-toxin or perfringolysin O pfoA Pore-forming toxin, cholesterol- CM: humans and animals Chm dependent cytolysin

Beta2-toxin cpb2 Putative pore-forming toxin No conﬁrmed association with Plasmid disease

TpeL tpeL Ras-speciﬁc mono- No conﬁrmed association with Plasmid glucosyltransferase disease

NetB netB Pore-forming toxin NE in poultry Plasmid

BecA, BecB becA/B Actin-speciﬁc ADP- GE in humans Plasmid ribosyltransferase

NetE netE Putative pore-forming toxin No conﬁrmed association with Plasmid disease

NetF netF Pore-forming toxin HE and NEC of dogs and foals Plasmid

NetG netG Putative pore-forming toxin No conﬁrmed association with Plasmid disease

NanI nanI Sialidase Accessory role Chm

Kappa-toxin colA Collagenase No conﬁrmed association with Chm disease

Mu-toxin nagH Hyaluronidase No conﬁrmed association with Chm disease

Lambda-toxin lam Protease No conﬁrmed association with Plasmid disease

a-clostripain ccp Cysteine Protease No conﬁrmed association with Chm disease

NanJ nanJ Sialidase No conﬁrmed association with Chm disease

Delta-toxin cpd Pore-forming toxin No conﬁrmed association with Plasmid disease

Chm, denotes a chromosomal gene location; CM, clostridial mynecrosis; C, colitis; E, enteritis; FP, food poisoning; GI, non-food borne gastrointestinal disease; NE, necrotising enteritis; ED, enteric disease; ET, enterotoxaemia; GE, gastroenteritis; HE, haemorrhagic enteritis; NEC, necrotising enterocolitis).

toxins, intracellular toxins, and hydrolytic enzymes4. Membrane comprise the largest toxin category and function to disrupt mem- damaging toxins such as alpha-toxin are enzymes that damage target brane permeability and ion transport by inserting into the mem- cell membranes through their ability to breakdown the constituents brane and forming a permeable channel or pore20. This category of the mammalian cell membrane19. The pore-forming toxins includes toxins such as perfringolysin O, beta-toxin, CPE, NetB and

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epsilon-toxin. Intracellular toxins, such as TpeL, BEC and iota-toxin, extracellular enzymes to cause a myriad of diseases. Note that the are internalised into target host cells where they act to disrupt the primary function of these toxins is most likely not to cause disease, cellular cytoskeleton21. Hydrolytic enzymes, such as sialidases and but to provide nutrients for the growth of C. perfringens cells, which hyaluronidases, are secreted by C. perfringens and degrade surface have limited capability to synthesise amino acids and essential associated glycans or glycoproteins4,22. These enzymes are not co-factors. Many of the toxins that are crucial contributors to disease, essential for disease; however, they may still contribute to the for example NetB, are encoded on conjugative plasmids10, and overall virulence of the bacterium23. therefore may be readily disseminated to other strains. Recent findings support the theory that C. perfringens strains have a tight Recent toxin discoveries association with the species in which they cause disease; for exam- In recent years, the number of characterised C. perfringens toxins ple, NetB-producing strains in birds and NetF-producers in foals and has increased significantly. The latest editions to the C. perfringens dogs. Just about everywhere we look, if there is an unclassified arsenal are the six novel toxins or putative toxins: NetB, BEC, TpeL, disease from which lots of C. perfringens cells can be isolated, the NetE, NetF and NetG. chances are good that another toxin is waiting to be discovered ... stay tuned! NetB is a beta-barrel pore-forming toxin and, like many other C. perfringens toxins, it is encoded on large conjugative plasmids24,25. Since its discovery, NetB-encoding plasmids have been identified in Acknowledgements many avian necrotic enteritis isolates; and netB deletion studies SAR-M is the recipient of an Australian Postgraduate Scholarship. have indicated that NetB toxin, is required for the development of necrotic enteritis in chickens26. References BEC is a novel binary toxin, composed of two distinct components, 1. McClane, B. et al. (2013) Clostridium perfringens.InFood microbiology: funda- BECa and BECb and it appears to function in a similar fashion to iota- mentals and frontiers, Fourth edn, pp. 465–489. ASM Press, Washington, DC. toxin15, with the BECa component having actin-specific ADP-ribo- 2. McClane, B.A. (2014) Clostridium perfringens.InEncyclopedia of Toxicology, Third edn (Wexler, P., ed.), pp. 987–988. Academic Press. syltranferase activity. BEC was discovered after two unrelated food 3. Uzal, F.A. et al. (2014) Towards an understanding of the role of Clostri- poisoning outbreaks that were caused by C. perfringens strains that dium perfringens toxins in human and animal disease. Future Microbiol. 9, did not encode CPE; the toxin typically associated with human food 361–377. doi:10.2217/fmb.13.168 poisoning15. Further studies showed that these strains produced a 4. Petit, L. et al. (1999) Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 7, 104–110. doi:10.1016/S0966-842X(98)01430-9 novel binary toxin, designated as BEC, suggesting that this new toxin 5. Uzal, F.A. et al. (2010) Clostridium perfringens toxins involved in mammalian was responsible for the enteric symptoms observed during these veterinary diseases. Open Toxinology J. 2,24–42. doi:10.2174/1875414701003 020024 outbreaks15. 6. Brynestad, S. et al. (2001) Enterotoxin plasmid from Clostridium perfringens is – TpeL is a member of the clostridial monoglycosyltransferase toxin conjugative. Infect. 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Annu. Rev. Micro- biol. 52, 333–360. doi:10.1146/annurev.micro.52.1.333 forming toxin NetF and the putative pore-forming toxins NetE and 12. Brynestad, S. and Granum, P.E. (2002) Clostridium perfringens and foodborne NetG, which were shown to be encoded on plasmids in isolates infections. Int. J. Food Microbiol. 74,195–202. doi:10.1016/S0168-1605(01) 00680-8 causing necrotizing gastroenteritis and necrotizing enterocolitis in 13. Bannam, T.L. et al. (2011) Necrotic enteritis-derived Clostridium perfringens 28 fi foals and dogs . There is a signi cant association between NetF- strain with three closely related independently conjugative toxin and antibiotic producing strains and enteric disease in these animals; however, the resistance plasmids. MBio 2, e00190-11. doi:10.1128/mBio.00190-11 function of NetE and NetG in disease remains to be investigated28. 14. Hughes, M.L. et al. (2007) Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative. J. Bacteriol. 189,7531–7538. doi:10.1128/JB.00767-07 15. Yonogi, S. et al. (2014) BEC, a novel enterotoxin of Clostridium perfringens Conclusion found in human clinical isolates from acute gastroenteritis outbreaks. Infect. Immun. 82, 2390–2399. doi:10.1128/IAI.01759-14 The production of toxins is essential for C. perfringens-mediated 16. Nagahama, M. et al. (2015) Recent insights into Clostridium perfringens beta- disease and this bacterium utilises an arsenal of different toxins and toxin. Toxins (Basel) 7, 396–406. doi:10.3390/toxins7020396

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17. Katayama, S. et al. (1996) Genome mapping of Clostridium perfringens strains 27. Chen, J. and McClane, B.A. (2015) Characterization of Clostridium perfringens with I-CeuI shows many virulence genes to be plasmid-borne. Mol. Gen. Genet. TpeL toxin gene carriage, production, cytotoxic contributions, and trypsin sen- 251,720–726. sitivity. Infect. Immun. 83, 2369–2381. doi:10.1128/IAI.03136-14 18. Miyamoto, K. et al. (2006) Complete sequencing and diversity analysis of the 28. Gohari, I.M. et al. (2015) A novel pore-forming toxin in type A Clostridium perfrin- enterotoxin-encoding plasmids in Clostridium perfringens type A non-food- gens is associated with both fatal canine hemorrhagic gastroenteritis and fatal borne human gastrointestinal disease isolates. J. Bacteriol. 188, 1585–1598. foal necrotizing enterocolitis. PLoS One 10, e0122684. doi:10.1371/journal.pone. doi:10.1128/JB.188.4.1585-1598.2006 0122684 19. Sakurai, J. et al. (2004) Clostridium perfringens alpha-toxin: characterization and mode of action. J. Biochem. 136,569–574. doi:10.1093/jb/mvh161 Biographies 20. Popoff, M.R. (2014) Clostridial pore-forming toxins: powerful virulence factors. Anaerobe 30,220–238. doi:10.1016/j.anaerobe.2014.05.014 Sarah A Revitt-Mills is a PhD student in the Department of 21. Sakurai, J. et al. (2009) Clostridium perfringens iota-toxin: structure and function. Microbiology at Monash University and is studying conjugative toxin Toxins (Basel) 1,208–228. doi:10.3390/toxins1020208 plasmid biology as part of her PhD studies. 22. Zukaite,_ V. and Biziulevicius, G.A. (2000) Physico-chemical and catalytic properties of Clostridium perfringens hyaluronidase: an update. Anaerobe 6, 347–355. doi:10.1006/anae.2000.0356 Julian I Rood is a Professor of Microbiology at Monash University 23. Chiarezza, M. et al. (2009) The NanI and NanJ sialidases of Clostridium perfrin- and has led the ﬁeld in clostridial conjugation mechanisms for gens are not essential for virulence. Infect. Immun. 77, 4421–4428. doi:10.1128/ IAI.00548-09 many years. He also studies bacterial pathogenesis, predominantly 24. Keyburn, A.L. et al. (2008) NetB, a new toxin that is associated with avian necrotic mechanisms utilised by the human and animal pathogen, Clostrid- ﬁ enteritis caused by Clostridium per ngens. PLoS Pathog. 4, e26. doi:10.1371/ ium perfringens, and the sheep pathogen, Dichelobacter nodosus. journal.ppat.0040026 25. Parreira, V.R. et al. (2012) Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative Vicki Adams is a Research Fellow in the Department of Microbi- plasmids. PLoS One 7, e49753. doi:10.1371/journal.pone.0049753 ology at Monash University and has studied mobile genetic elements 26. Keyburn, A.L. et al. (2010) Association between avian necrotic enteritis and found in the clostridia, in addition to the biology of large conjugative Clostridium perfringens strains expressing NetB toxin. Vet. Res. 41, 21. doi:10.1051/vetres/2009069 toxin plasmids of Clostridium perfringens.

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